Sugarcane (Saccharum spp.) is a grassy plant belonging to the botanic family Poaceae, originating from Southeast Asia, from the large central region of New Guinea and Indonesia (Daniels & Roach, 1987, Sugarcane improvement through breeding p. 7-84). It is one of the most important plant species cultivated in tropical and subtropical regions, with an area exceeding 23 million hectares distributed over 121 countries (FAO Statistical Yearbook 2012 p. 233).
The sugarcane cultivated area is on the increase, and it is a source of raw materials for the production of sugar, wine, molasses, ruin, cachaça (the national distilled of Brazil) and fuel ethanol. The bagasse remaining after milling the sugarcane can be used for balering and supply of heat energy, used in mill, and electricity, which is typically sold to the consumer electrical grid, and as a base for producing ethanol. Therefore, the sugarcane agroindustry, responsible for generating millions of jobs in the area, is of major social importance, generating revenue through the export of sugar and ethanol and by the rational use of the plant biomass.
More recently, with increased concern over global warming and use of alternative sources to fossil fuels (biofuels), world interest in sugarcane has increased significantly.
Due to the economic and social importance of sugarcane, major research efforts are noted, aimed at defining better agricultural practices for cultivation and improved quality of the varieties cultivated.
In this regard, conventional plant breeding methods have proven limited for the introduction of genes and traits of interest in different varieties of commercial interest.
Due to this difficulty and the growing need to incorporate desirable traits, such as, for example, increased productivity, tolerance to insects, pathogen and herbicides, resistance to abiotic stresses, etc., molecular biology methods have been used to manipulate sugarcane.
The genetic engineering of plants involves the transfer of genes of interest into the plant cells, such that a fertile and agronomically superior progeny that maintains and expresses in a stable manner the exogenous gene can be obtained. Accordingly, one of the options is the use of in vitro cultivation techniques.
One of the techniques of in vitro cultivation is somatic embryogenesis, which consists of the production of embryos from an isolated cell or a small group of cells which, by means of in vitro cultivation will give rise to somatic embryos. In this case, structures similar to zygotic embryos develop from somatic cells, following a sequence of characteristic stages of zygotic embryogenesis, giving rise to a plant, without the fusion of gametes (Jimenez. 2001. Regulation of in vitro somatic embryogenesis with emphasis on the role of endogenous hormones. Revista Brasileira de Fisiologia Vegetal, v. 13, p. 196-223).
According to Guerra et al., Embriogênese somática e sementes sintéticas. In: Tones et al. (Eds.). 1999. Cultura de tecidos e transformação genética de plantas. Brasilia: Embrapa, v.2, p. 533-568, a striking characteristic of somatic embryos is the presence of a closed vascular system, without vascular connection with the tissues of the initial explant. This characteristic coupled with its bipolarity (presence of shoot and root apices), enables the distinction between the processes of embryogenesis and organogenesis (Falco et al. 1996. Histological characterization of in vitro regeneration of Saccharum sp. Revista Brasileira de Fisiologia Vegetal, v.9, p. 93-97).
According to Suprasanna et al. (2005. Regulation of somatic embryogenesis by plant growth regulators in sugarcane. Sugar tech, v. 7, p. 123-128), the use of somatic embryogenesis in the cultivation of sugarcane has two main objectives: the development of a reproducible method for the fast propagation of plants and the achievement of an efficient system of regeneration of somatic embryos used for genetic transformation.
Various types of explants have been used in the embryonic process in sugarcane. According to Lakshmanan et al. (2006. Developmental and hormonal regulation of direct shoot organogenesis and somatic embryogenesis in sugarcane (Saccharum spp. Interspecific hybrids) leaf culture. Plant Cell Reports, v. 25, p. 1007-1015), almost all plant tissues give rise to embryogenic calluses, but the younger leaves (Chengalrayan & Gallo-Meagher. 2001. In vitro Cellular and Developmental Biology-Plant, Oxon, v. 37, p. 434-439; Lakshmanan et al. 2006. Developmental and hormonal regulation of direct shoot organogenesis and somatic embryogenesis in sugarcane (Saccharum spp. Interspecific hybrids) leaf culture. Plant Cell Reports, v. 25, p. 1007-1015; Snyman et al. 2011. Applications of in vitro culture systems for commercial sugarcane production and improvement. In vitro Cellular and Developmental Biology Plant, v. 47, p. 234-249, 2011) and developing inflorescences (Gallo-Meagher et al. 2000. Thidiazuron stimulates shoot regeneration of sugarcane embryogenic callus. In vitro Cellular and Developmental Biology Plant, v. 36, p. 37-40); Desai et al. 2004. Current Science, Bangalore, v. 87, p. 764-768), are very prolific and are preferred target tissues for fast production of embryogenic calluses.
Somatic embryogenesis is initiated by adding growth regulators to the culture medium and, among these, the auxins stand out as the class of growth regulators most used in the embryonic process (Cooke et al. 1993. The role of auxin in plant embryogenesis. The Plant Cell, v. 5, p. 1494-1495, 1993). The 2,4D (2,4-dichlorophenoxyacetic acid) is the growth regulator most used in the induction process of somatic embryogenesis in sugarcane.
The conversion of the somatic embryos in plants is the final phase of the process of somatic embryogenesis. Regeneration generally occurs in a medium devoid of growth regulators and in the presence of light (Garcia et al. 2007. In vitro morphogenesis patterns from shoot apices of sugarcane are determined by light and type of growth regulator. Plant Cell, Tissue and Organ Culture, v. 90, p. 181-190; Watt et al. 2009. In vitro minimal growth storage of Saccharum spp. Hybrid (genotype 88H0019) at two stages of direct somatic embryogenic regeneration. Plant Cell, Tissue and Organ Culture, v. 96, p. 263-271; Suprasana, et al. 2010. Profiling of culture-induced variation in sugarcane plants regenerated via direct and indirect somatic embryogenesis by using transposon-insertion polymorphism. Sugar Tech, v.12, p. 26-30; Van Der Vyver, C. 2010. Genetic transformation of the euploid Saccharum officinarum via direct and indirect embryogenesis. Sugar tech, v. 12, p. 21-25; Basnayake et al. 2011. Embryogenic callus proliferation and regeneration conditions for genetic transformation of diverse sugarcane cultivars. Plant Cell Reports, v. 30, p. 439-448), however, this process may be improved by using different regulators (Ali et al. 2008. An efficient protocol for large scale production of sugarcane through micropropagation. Pakistan Journal of Botany, v.40, p. 139-149; Nieves et al. 2008. Effect of exogenous arginine on sugarcane (Saccharum sp.) somatic embryogenesis, free polyamines and the contents of the soluble proteins and proline. Plant Cells, Tissue and Organ Culture, v. 95, p. 313-320; Kaur & Gosal. 2009. Callus desiccation enhances somatic embryogenesis and subsequent shoot regeneration in sugarcane. Indian Journal of Biotechnology, v. 8, p. 332-334; Goel et al. 2010. In vitro morphogenesis in leaf sheath explants of sugarcane hybrid var. CoS 99259. Sugar Tech, v. 12, p. 172-175; Wamaitha et al. 2010. Thidiazuron-induced rapid shoot regeneration via embryo-like structure formation from shoot tip-derived callus culture of sugarcane. Plant Biotechnology, v. 27, p. 365-368).
Recombinant DNA technology has enabled the isolation of genes and the stable insertion into a host genome (Quecini & Vieira.2001. Plantas transgênicas. In: Serafini et al. (Ed.). Biotecnologia na agricultura e na agroindústria. Guaíba: Agropecuária, p. 278-331). This technique, also called genetic transformation, may be defined as the controlled introduction of nucleic acids (DNA) in a host genome, excluding the introduction by fecundation. It is a more controlled process, where normally only the defined DNA fragment is introduced into the host genome, or receptor genome, and must be integrated thereto (Brasileiro & Dusi. 1999. Transformação genética de plantas. In: Torres et al. (Ed.) Cultura de tecidos e transformação genética de plantas. Brasilia: EMBRAPA-SPI/EMBRAPA-CNPH, 863p, v.2). The stable insertion of these molecules into a host genome gives rise to an individual identical to the receptor of the recombinant molecule, but with the addition of a new and particular characteristic (Quecini & Vieira, 2001, above).
There are various techniques of genetic transformation of plants grouped into two categories: indirect and direct transfer of genes. Indirect transfer is the one in which the exogenous DNA is inserted into the genome by the action of a biological vector, whereas direct transfer is based on physical-biochemical processes.
Indirect transformation is chiefly based on the system mediated by bacteria of the genus Agrobacterium and has been the most used method to obtain transgenic plants. Agrobacterium tumefaciens and A. rhizogenes are phytopathogenic soil bacteria, grain negative, belonging to the Rhizobiaceae family, which cause diseases in dicot, known as crown galls and hairy root, respectively. In this plant-pathogen interaction there occurs a process of natural transfer of genes between the agrobacteria and the plant cell, when bacterial DNA fragments (T-DNA) are transferred into the plant cell, integrating the nuclear genome (Ream & Gelvin. 1996. Crown gall: Advances in understanding interkingdom gene transfer. Saint Paul: APS Press, 148p). In its natural form, the bacteria transfers the T-DNA (“transferred DNA”), which is the part of the bacterial plasmid called Ti (“tumor-inducing”), and this integrates the genome of the infected plant cells. In the T-DNA fragment that is transferred to the plant cell are the genes involved in the phytohormone-constituting biosynthesis (auxins and cytokines) that alter the program of normal development of the infected tissue, causing the formation of the tumor. Additionally, it also contains oncogenes for the synthesis of sugars and amine acids called opines, which are responsible for the survival of the bacteria, which use them as a source of carbon and nitrogen (Oger et al. 1997. Genetically engineered plants producing opines alter their biological environment. Nature Biotechnology, New York, v. 15, p. 369-372).
Repeated ends of 25 base pairs (bp) on the right and left edges delimit the T-DNA and are essential for the transfer thereof (Wang et al. 1984. Cell, Cambridge, v. 38, p. 455-462). Phenolic compounds released by the wounded plant tissues activate specific regions (vir), initiating the T-DNA transfer process to the plant cell (Stachel et al. 1985. Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature, London, v. 318, p. 624-629). The Agrobacterium also has chromosomal genes (chv) that assure the link between the bacterial and host cells, enabling the formation of the passage pore of the T-strand complex (Sheng & Citovsky. 1996. Agrobacterium-plant cell DNA transport: have virulence proteins, will travel. The Plant Cell, Baltimore, v.8, p. 1699-1710).
The virulence region, called vir region is responsible for the transfer process, and the induction process and transfer of strand—T is controlled by the coordinated expression of this region. The virA locus encodes a membrane protein that perceives the presence of metabolites of the wounded plant (acetosyringone). After bonds to acetosyringone , the “activated” VirA protein modifies the VirG protein, which is also expressed constitutively, but on a lower scale through phosphorylation thereof. The phosphorylated VirG protein is responsible for inducing the transcription of the entire vir region. To form the T-strand, the operon virD encodes endonucleases capable of recognizing and cleaving inside the 25 bp which delimit the region-T. The transfer of the T-strand is polar, always from right to left. The T-strand is transferred to the plant cell in the form of a single strand, protected in 5′portion of the molecule by the protein VirD2, and throughout the T-strand by protein VirE2 (Zambryski. 1992. Chronicle from the Agrobacterium-plant cell DNA transfer story. Annual Review of Plant Physiology and Plant Molecular Biology, Palo Alto, v. 43, p. 465-490). The T-DNA released is protected by bonds throughout the single strand by the protein VirE2, which is also responsible for the structural organization of the strand during the path between the bacterial cell and the plant cell. Encoded proteins by the locus virB assure the passage through the membrane of the bacteria, by the formation of passage pore between the membrane and the cell wall (Zambryski, 1992, above).
The process of transfer may be split into two main steps: a bacterial step and an eukaryotic step that occurs in the plant cell (Zupan & Zambryski. 1995. Plant Physiology, Rockville, v. 107, p. 1041-1047). The bacterial step includes the production and export of a functional vector containing the genetic information of the T-DNA (Tinland. 1996. The integration of T-DNA into plant genomes. Trend in Plant Science, Kidlington, v. 1, p. 178-183). The eukaryotic step includes the recognition between the Agrobacterium and the host cell, the transduction of plant signals of pathogenesis and the activation of the vir genes (Sheng & Citovsky. 1996, above). Since the segment to be transferred is defined by its edges, the encoding region of the wild-type T-DNA may be replaced by any other DNA sequence without impairing its transfer from Agrobacterium to the plant. The replacement of the oncogenes by genes of interest, flanked by the edges of the T-DNA, provides an efficient system of obtaining transgenic plants (Brasileiro & Dusi. 1999, above).
The vectors used for the transformation via A. tumefaciens are called “disarmed”, that is, they do not have the oncogenes in their plasmid, but retain the virulence genes (vir region), located in the plasmid Ti (Ream & Gelvin. 1996. Crow gall: advances in understanding interkingdom gene transfer. Saint Paul: APS Press, 148p). These plasmid constructions have plant promoters and bacterial genes that confer resistance to antibiotics, making these markers efficient for the selection of cells or transformed plants. Therefore, A. tumefaciens is used as transformation vector, where the T-DNA fragment is eliminated and replaced by a gene of interest (Saciloto. 2003. Insertion of the PR5K gene in sugarcane with a view to inducing resistance to the Puccinia melanocephala rust fungus. 74p. Master's dissertation presented before the Luiz de Queiroz Superior School of Agriculture, University of São Paulo, Piracicaba), losing the capacity to cause tumors, but being capable of transferring the exogenous DNA. Explants inoculated with the disarmed strains have a regenerative capacity and large production percentage of transgenic plants (Brasileiro. 1998. Manual de transformação genética de plantas. Brasília: EMBRAPA, CENARGEN, 309p).
The complexity of the polyploid and aneuploid genome of sugarcane varieties (D'Hont & Glaszmann. 2005. Unraveling the genome structure of polyploids using FISH and GISH; examples of sugarcane and banana. Cytogenetic and Genome Research, Basel, v. 109, n. 1-3, p. 27-33), added to its relatively restrict genetic basis, imposes major difficulties to the application of conventional plant breeding techniques. Considered this situation, biotechnology may be applied in plant breeding programs to overcome or reduce some of the limitations of conventional approaches, and increase the productivity of sugarcane biofuel. For this, certain characteristics can be incorporated to the culture by way of genetic engineering through the genetic transformation of plants, to reduce the losses with biotic stresses associated with pests, plant weeds and diseases, and abiotic stresses related to drought, cold, salinity among others. Biotechnology can also make changes to optimize the content and the quality of sugar (Melotto-Passarin. (2009). Doctorate Thesis in Physiology and Biochemistry of Plants presented at the “Luiz de Queiroz” Superior School of Agriculture, University of São Paulo, Piracicaba, 148p.).
Sugarcane presents characteristics that make it an excellent plant for improvement through genetic transformation, such as its facility for regenerating plants from calluses in vitro (Heinz et al. 1997. Cell, tissue and organ culture in sugarcane improvement. In: Reinert & Bajaj (Ed.). Applied and fundamental aspects of plant cell, tissue and organ culture. Berlin: Springer Verlag, p. 3-17; Irvine 1984. The frequency of marker change in sugarcane plants regenerated from callus culture. Plant Cell, Tissue and Organ Culture, Dordrecht, v. 3, n. 3, p. 201-209; Chen et al. 1988. Control and maintenance of plant regeneration in sugarcane callus cultures. Journal of Experimental Botany, Oxford, v. 39, p. 251-261) and, its multiplication mode on commercial scale by vegetative propagation that enables distribution of stable transformants to the producers through seedlings (Gallo-Meagher & Irvine. 1996. Herbicide resistant transgenic sugarcane plants containing the bar gene. Crop Science, Madison, v. 36, it 5, p. 1367-1374). In contrast, it does not allow the use of zygotic embryo as target tissue in the transformation, contrary to maize, rice, wheat and other commercial cereal crops.
Over the last decade, various researches have been carried out to develop efficient methods of genetic transformation of sugarcane (Chen et al. 1987. Transformation of sugarcane protoplasts by direct uptake of a selectable chimeric gene. The Plant Cell Reports, New York, v.6, p. 297-301; Bower & Birch. 1992. The Plant Journal, Oxford, v. 2, n. 3, p. 409-416; Rathius & Birch, R. G. 1992. Stable transformation of callus from electroporated sugarcane protoplasts. Plant Science, Amsterdam, v. 82, p. 81-89; Smith et al. 1992. Transient expression of the coat protein of sugarcane mosaic virus in sugarcane protoplasts and expression in Escherichia coli. Archives of Virology, Vienna, v. 125, p. 15-23; Birch & Maretzki, A. 1993. Transformation of sugarcane. In: Bajaj, Y. P. S. (Ed.). Plant protoplasts and genetic engineering IV. Biotechnology in Agriculture and Forestry. Heidelberg: Springer-Verlag, v. 23, p. 348-360; Gambley et al. 1993. Microprojetile transformation of sugarcane meristems and regeneration of shoots expressing β-glucuronidase. The Plant Cell Reports, New York, v. 12, p. 343-346; Gambley et al. 1994. Australian Journal of Plant Physiology, Melbourne, v. 21, p. 603-612; Birch. 1997. Plant transformation: problems and strategies for practical application. Annual Review of Plant Physiology and Plant Molecular Biology, Palo Alto, v. 48, p. 297-326; Arencibia. 1998. An efficient protocol for sugarcane (Saccharum spp. L.) transformation mediated by Agrobacterium tumefaciens. Transgenic Research, New York, v. 7, p. 213-222; Elliott et al. 1998. Australian Journal of Plant Physiology, Melbourne, v. 25, p. 739-743; Enriquez-Obregon et al. 1998. Plant, Berlin, v. 206, p. 20-27; Manickavasagam et al. 2004. Agrobacterium-mediated genetic transformation and development of herbicide-resistant sugarcane (Saccharum species hybrids) using axillary buds. The Plant Cell Reports, New York, v. 23, it 3, p. 134-143). Different transformation techniques using electroporation (Rathius & Birch. 1992. Stable transformation of callus from electroporated sugarcane protoplasts. Plant Science, Amsterdam, v. 82, p. 81-89), treatment with polyethylenoglycol (PEG) (Chen et al. 1987. Transformation of sugarcane protoplasts by direct uptake of a selectable chimeric gene. The Plant Cell Reports, New York, v.6, p. 297-301), microprojectile bombardment (Franks & Birch 1991. Gene transfer into intact cells using microprojectile bombardment. Australian Journal of Plant Physiology, Melbourne, v. 18, p. 471-480) and Agrobacterium tumefaciens (Arencibia. 1998. An efficient protocol for sugarcane (Saccharum spp. L.) Transformation mediated by Agrobacterium tumefaciens. Transgenic Research, New York, v. 7, p. 213-222; Elliott et al. 1998. Agrobacterium-mediated transformation of sugarcane using GFP as a screenable marker. Australian Journal of Plant Physiology, v. 25, p. 739-743) were used to introduce marker genes in cells and cane calluses. The first transgenic cane cells were obtained following the transfer of DNA for protoplasts mediated by PEG (Chen et al. 1987. Transformation of sugarcane protoplasts by direct uptake of a selectable chimeric gene. The Plant Cell Reports, New York, v.6, p. 297-301).
The first attempts at transforming sugarcane using Agrobacterium tumefaciens, with or without virulence gene inhibitors and other treatments that improve the infection, had little success (Birch & Maretzki. (1993). Transformation of sugarcane. In: Bajaj, Y.P.S. (Ed.). Plant protoplasts and genetic engineering IV. Biotechnology in agriculture and forestry. Heidelberg: Springer-Verlag, v. 23, p. 348-360). However, Arencibia (1998. An efficient protocol for sugarcane (Saccharum spp. L.) Transformation mediated by Agrobacterium tumefaciens. Transgenic Research, New York, v. 7, p. 213-222) was capable of regenerating morphologically normal transgenic sugarcane plants following the co-cultivation of calluses with Agrobacterium tumefaciens strains LBA4404 and EHA101. Almost simultaneously, Enriquez-Obregon et al. (1998, Plant, Berlin, v. 206, p. 20-27) reported the production of cane plants resistant to the commercial herbicide BASTA (active component phosphinothricine). However, few laboratories managed to repeat these pioneer works of agrobacteria in sugarcane at the time following the publications.
With suitable handling and control of the in vitro culture conditions, considering the best age, type and stage of the embryonic culture, and also the improvement of the virulence of the A. tumefaciens strain demonstrated enhanced transformation efficiency. This natural transformation model presents advantages of transferring relatively long DNA segments with no re-arrangement, integrating a small number of copies of the transgenes in genome sites with high expression rate, being a simple and low-cost methodology (Melotto-Passarin, 2009, above).
Besides producing good agricultural products, genetic transformation technology also offers the possibility of studying thousands of plant genes (with known and unknown functions) which have been identified by the countless genome programs conducted throughout the world over recent years (Dong et al. 2005. Plant Physiology, Rockville, v. 139, p. 610-618).
Although the transformation methods mediated by Agrobacterium are used for the genetic manipulation of sugarcane, it is broadly recognized by those skilled in the art that the efficiency and reproducibility of the methodologies also constitute challenges to be overcome. In any technology for transforming plants, there are multiple factors that influence the success of the transfer of a gene of interest in plants, and its subsequent stable integration and expression. One of the aspects that might affect the transformation success is the growth of Agrobacterium in relation to transformed plant cells. It is known that if there is exacerbated growth of Agrobacterium, the chances of regenerating plants from transformed cells decreases. This may be due to the necrosis induced by Agrobacterium, in a process in which the tissue firstly undergoes a process of oxidation and browning and subsequently dies.
The inoculation of a plant tissue with Agrobacterium is, in itself, a process that unleashes hypersensitivity responses, resulting in a low survival rate of the tissue. Therefore, the planning of a suitable artificial environment to minimize the damages due to the interaction of the plant tissue with Agrobacterium is critical for the success of genetic transformation experiments.
Document WO 200109302 discloses control of the growth of Agrobacterium as a form of improving the efficiency of transformation, through the use of inhibitor agents during inoculation and co-culture of Agrobacterium with the plant tissue. Preferred inhibitor agents are compounds containing heavy metals, such as silver nitrate or silver thiosulphate, antibiotics such as carbenicillin and a combination of antibiotics and a clavulanic acid, such as augmentin or timetin.
Document U.S. Pat. No. 6,323,396 discloses a process for obtaining transgenic plants using mutant Agrobacterium deficient in the biosynthesis of vital specific biomolecules. This will enable the maintenance of a controlled systemic infection of the tissue to be transformed for long periods, increasing the likelihood of success in the infection. The Agrobacterium is eliminated by the omission of these nutrients from the incubation medium.
Document WO2010151634 discloses the co-cultivation in desiccant conditions, in the absence of culture medium, mentioning that this beneficially reduces the necrosis/apoptosis of the inoculated plant tissue, besides improving the subsequent cellular survival during the selection and regeneration steps which typically follow on from the co-cultivation step.
Document WO 98/54961 discloses antinecrotic treatments including cultivation in a necrosis inhibitor medium containing an ethylene or ethylene biosynthesis inhibitor, heat shock treatment of the cells or tissues before co-cultivation with Agrobacterium and transformation of the cells of grasses, chiefly maize, with genetic sequences such as p35, iap and dad-1.
Document WO 01/44459 describes agents that inhibit the activity or production of enzymes associated with the browning of plant tissues during the transformation mediated by Agrobacterium, such as polyphenol oxidase (PPO) and peroxidase (POD), metal chelators necessary for enzyme activity, and agents containing sulfhydryl (e.g. L-cysteine, cysteine, DTT, ascorbic acid, sodium thiosulfate and glutathione). The inhibited enzymes include oxidase (PPO) and peroxidase.
In view of this problem in the state of the art, the present invention provides a method of genetic transformation that contributes to the genetic plant breeding programs and functional studies of new genes, including those with complex multigene characteristics, by establishing new culture conditions during the co-culture of Agrobacterium with the plant tissue to be transformed, resulting in an improvement compared with existing methods. The inventors believe that the method presented herein, due to the advantages and unexpected effects obtained, may contribute to minimize the intrinsic limitations of the genetic breeding of plants of interest, including, but not limited to sugarcane.